Many bioactive materials such as medicines are in a form of various enantiomer, and only a specific enantiomer has the desired efficacy, and the remnant enantiomers causes serious undesirable effect. In aspects of safety and bioactivity, only single enantiomer must be produced, and thus many researches on the synthesis of enantio-pure bioactive material can be processed.
Enantiopure epoxides and vicinal diols are versatile synthetic intermediates for the preparation of enantiopure bioactive compounds such as pharmaceutical compounds, pesticides, and functional foods (Grogan, et al., FEMS Microbiol. Lett., 141:239-243, 1996; Arahira, et al., Eur. J. Biochem., 267:2649-2657, 2000), because the compounds has excellent reactivity and can induce the various reactions.
Particularly, the enantiopure epoxides can be prepared by using the chiral chemical catalysts and enzymes, and only single enantiomer is prepared by performing the selective hydrolysis with epoxide hydrolase to each enantiomer in racemic epoxide substrates. The method can be used commercially in the near future, because it can change inexpensive racemic substrate to enantiopure epoxide having higher added value. The epoxide hydrolase hydrolyzes only (R) or (S)-enantiomer among racemic epoxide substrate with enantio-selectivity to diol and leave the other type of enantiomer, so as to produce enantiopure epoxide. In addition, the enantioselectivity of epoxide hydrolase to (R) or (S)-enantiomer depends on microorganisms and substrate structure.
Epoxide hydrolases (EHase; EC 3.3.2.3) are ubiquitous enzymes that have been isolated from a wide variety of sources such bacteria, yeast, fungi, insect, plant and mammalian (Weijers, et al., J. Mol. Catal. B Enzym., 6:199-214, 1999; Archelas, & Furstoss, Curr. Opin. Chem. Biol., 5:112-119, 2001). Due to the potential application in the production of enantiopure epoxides by kinetic resolution of enantioselective EHase, several EHases have been developed (Tokunaga, et al., Science, 277:936-938, 1997).
However, the limited number of enantioselective EHases demands studies to explore new enantioselective EHases for the production of enantiopure epoxides in pharmaceutical industries.
Most EHases are members of the α/β hydrolase family which includes proteases, lipases, esterases, dehalogenases, and peroxidases (Nardini, & Dijkstra, Curr. Opin. Struct. Biol., 9:732-737, 1999; Rick, et al., J. Am. Chem. Soc., 121:7417-7418, 1999). α/β domains consist of a central, parallel or mixed β sheet surrounded by α helices. These enzymes characteristically employ a two-step mechanism in which a catalytic nucleophile of the enzyme attacks a polarized electrophile substrate of the covalent intermediate subsequently hydrolyzed (Yamada, et al., J. Biol. Chem., 275:23082-23088, 2000). The conserved catalytic triad of α/β hydrolase fold enzymes consists of a nucleophilic residue (Asp or Ser), an acidic residue (Asp or Glu) and a conserved histidine residue. The nucleophile fits the conserved amino-acid-sequence motif, Sm-X-Nu-Sm (Sm=small residue, X=any residue and Nu=nucleophile). Another conserved amino acid sequence is the HGXP motif containing the oxyanion hole of the enzyme (Ollis, et al., Protein Eng., 5:197-211, 1992).
However, the conservation in the primary sequence among EHases is limited only in 2 or 3 amino acids of the critical regions, leading to make the screening by homology search difficult.